Linear Velocity Of Mobile Phase Research Articles

Study of the column efficiency using gradient elution based on Van Deemter plots

Performance of chromatographic columns is of major importance in the development of more efficient separation methods. So far, a common practice is to study the column behavior in isocratic elution by modifying the flow rate and fitting the theoretical plate height values versus the mobile phase linear velocity, according to the Van Deemter equation. In this work, an approach is presented to extend the measurement of efficiency to linear gradient elution, where the mean retention factor is kept constant at each assayed flow. This avoids a possible source of uncertainty due to the change in the distribution equilibria profile, and makes the mean interactions with the stationary phase in gradient elution similar to those in isocratic elution. The approach was applied to Zorbax Eclipse XDB C18 and Chromolith SpeedROD C18 columns, using four sulphonamides as probe compounds. The results are compared with those obtained in isocratic elution, and gradient elution where the gradient program is kept constant while the flow is varied, giving rise to changes in the mean retention factor at each flow rate. The mean compression factor obtained experimentally was higher than expected, with mean values of 0.98 and 1.02 for the Zorbax and Chromolith columns, whereas the predicted values were 0.87 and 0.92, respectively. Better efficiencies were obtained at lower flow and higher gradient slope. The resolution shows similar values in isocratic and gradient elution when the mean retention factor is kept constant. When the gradient slope is constant, the resolution is usually smaller, although it improves at higher flows.

Thin-layer chromatogram development with a moving pipette delivering the mobile phase onto the surface of the adsorbent layer

The new approach to the development of thin-layer chromatograms is presented. For the first time we show flexible mobile phase dosage onto the surface of the adsorbent layer by moving pipette combined with precise syringe pumps. The pipette is driven into movement by computer controlled 3D machine (modified 3D printer mechanism). Delivery of the mobile phase to the adsorbent layer is equal to or lower than that of conventional development. Therefore chromatograms can be developed with optimal mobile phase velocity, adjusted to its absorption rate by the adsorbent layer. Under such conditions there is no excess of eluent on the surface of the adsorbent layer so higher performance of the chromatographic system can be obtained. Moreover chromatograms can be developed with constant linear mobile phase velocity and therefore the relationships the plate height vs. mobile phase linear velocity obtained with planar chromatography driven by capillary forces are investigated and reported. In addition the contribution of starting spot variance in total peak variance and the influence of narrowing of starting spots on performance of the chromatographic system have been studied. The results confirm a very significant starting spot variance contribution to total peak variance and consequently considerable influence of starting spot size on plate height of the separation system, when chromatogram is developed on a short distance.In the paper the advantages and disadvantages of the prototype device and its possible application are discussed.

Schinus terebinthifolius countercurrent chromatography (Part III): Method transfer from small countercurrent chromatography column to preparative centrifugal partition chromatography ones as a part of method development

Countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) are support free liquid-liquid chromatography techniques sharing the same basic principles and features. Method transfer has previously been demonstrated for both techniques but never from one to another. This study aimed to show such a feasibility using fractionation of Schinus terebinthifolius berries dichloromethane extract as a case study. Heptane − ethyl acetate − methanol −water (6:1:6:1, v/v/v/v) was used as solvent system with masticadienonic and 3β-masticadienolic acids as target compounds. The optimized separation methodology previously described in Part I and II, was scaled up from an analytical hydrodynamic CCC column (17.4mL) to preparative hydrostatic CPC instruments (250mL and 303mL) as a part of method development. Flow-rate and sample loading were further optimized on CPC. Mobile phase linear velocity is suggested as a transfer invariant parameter if the CPC column contains sufficient number of partition cells.

Microwave-assisted grafting polymerization modification of nylon 6 capillary-channeled polymer fibers for enhanced weak cation exchange protein separations

A weak cation exchange liquid chromatography stationary phase (nylon-COOH) was prepared by grafting polyacrylic acid on to native nylon 6 capillary-channeled polymer (C-CP) fibers via a microwave-assisted radical polymerization. To the best of our knowledge, this is the first study of applying microwave-assisted grafting polymerization to affect nylon material for protein separation. The C-CP fiber surfaces were characterized by attenuated total reflection (ATR) infrared spectroscopy and scanning electron microscope (SEM). The anticipated carbonyl peak at 1722.9 cm−1 was found on the nylon-COOH fibers, but was not found on the native fiber, indicating the presence of the polyacrylic acid on nylon fibers after grafting. The nylon-COOH phase showed a ∼12× increase in lysozyme dynamic binding capacity (∼12 mg mL−1) when compared to the native fiber phase (∼1 mg mL−1). The loading capacity of the nylon-COOH phase is nearly independent of the lysozyme loading concentration (0.05–1 mg mL−1) and the mobile phase linear velocity (7.3–73 mm s−1). The reproducibility of the lysozyme recovery from the nylon-COOH (RSD = 0.3%, n = 10) and the batch-to-batch variability in the functionalization (RSD = 3%, n = 5) were also investigated, revealing very high levels of consistency. Fast baseline separations of myoglobin, α-chymotrypsinogen A, cytochrome c and lysozyme were achieved using the nylon-COOH column. It was found that a 5× increase in the mobile phase linear velocity (7.3-to-36.5 mm s−1) had little effect on the separation resolution. The microwave-assisted grafting polymerization has great potential as a generalized surface modification methodology across the applications of C-CP fibers.

Capillary methacrylate-based monoliths by grafting from/to γ-ray polymerization on a tentacle-type reactive surface for the liquid chromatographic separations of small molecules and intact proteins

Capillary methacrylate-based monoliths were prepared for the high performance liquid chromatography (HPLC) separation of both small molecules and large biomolecules. An efficient grafting from/to synthetic approach was adopted introducing a network of activated sites in the inner wall surface using the new silanization agent (N-trimethoxysilylpropyl)-polyethylenimine. Copolymerization of lauryl methacrylate monomer and 1,6-hexanediol dimethacrylate cross-linker in the presence of porogenic solvents was obtained under continuous γ-ray exposure with high conversion yield.The morphology and porous structure of the resulting monoliths have been investigated by Scanning Electron Microscopy (SEM) and 1H NMR cryoporosimetry. By chromatographic investigation, the new capillary columns attested high kinetic performance (with efficiency larger than 100,000 theoretical plate/m for small molecules at optimum mobile phase linear velocity of about 0.5mm/s) and also excellent mechanical stability and repeatability.The new methacrylate-based monolithic capillary columns have been successfully employed for efficient reversed-phase separation of intact proteins and peptides.

Pressure-Driven Chromatographic Separation Modes in Self-Enclosed Integrated Nanocapillaries.

This study presents pressure-driven chromatographic separation characteristics of integrated nanofluidic channels (nanocapillaries) featuring distinct cross-sectional geometries: cylindrical, triangular, and rectangular profiles. Cylindrical and triangular nanocapillaries are self-enclosed robust conduits realized through standard semiconductor processing techniques using low-resolution photolithography. Specifically, capillaries in nominal radius 300 and 500 nm have been investigated for chromatographic separation in comparison to 750 nm deep nanoslits as well as triangular capillaries featuring an inscribed circle about 500 nm in radius. Chromatograms have been obtained from 10 mm long nanocapillaries under various modes: normal- and reversed-phase, ion-valence, and hydrodynamic chromatography. The van Deemter plots based on the linear mobile phase velocity for 300 nm radius capillaries and 750 nm deep slits show excellent agreement with the plate heights theoretically predicted. The minimum plate heights achieved are typically below 2 μm and the theoretical plate numbers are in the order of 105 plates/m for the most chromatography modes investigated in the pressure range up to 100 psi. A comparatively high resolving power is achieved with cylindrical nanocapillaries especially those 300 nm in radius. Self-enclosed robust nanocapillaries demonstrated here could facilitate a pressure-driven chromatographic analysis of extremely low-volume samples (e.g., single cell).

Effect of operating parameters on a centrifugal partition chromatography separation

Centrifugal partition chromatography (CPC) is the branch of countercurrent chromatography (CCC) that works with single axis hydrostatic columns with rotary seals. The hydrodynamic of the liquid stationary phase-liquid mobile phase equilibrium in the CPC chambers has been studied theoretically and with specially designed CPC columns. In this work, we selected a simple analytical separation (no loading study) of three test solutes, coccine red, coumarin and carvone, with a commonly used heptane/ethyl acetate/methanol/water 1:1:1:1v/v biphasic liquid system and two different rotors: a commercially available 30-mL CPC instrument and a 80-mL prototype rotor designed for productivity. We fully studied this separation in many possible practical operating conditions of the two rotors, aiming at a generic column characterization. The rotor rotation was varied between 1000 and 2800rpm, the aqueous mobile phase flow rate was varied between 1 and 22mL/min with the 30-mL rotor and 10 and 55mL/min with the 80-mL rotor, the upper limits being mechanical constraints and some liquid stationary phase remaining in the rotor. The variations of Sf, the volume ratio of stationary phase in the rotor, were studied versus mobile phase flow rate and rotor rotation speed. A maximum mobile phase linear velocity was found to depend on the centrifugal field for the 30-mL rotor. This maximum velocity was not observed with the 80-mL rotor. Studying the changes in coumarin and carvone peak efficiencies, it is established that the number of cells required to make one theoretical plate, i.e. one chromatographic exchange, is minimized at maximal rotation speed and, to a lesser extent, at high mobile phase flow rate (or linear velocity). Considering the throughput, there is evidence of an optimal flow rate depending on the rotor rotation that is not necessarily the highest possible.

Prediction of Peak Shape and Characterization of Column Performance in Liquid Chromatography as a Function of Flow Rate

Traditionally, column performance in liquid chromatography has been studied using information from the elution of probe compounds at different flow rates through van Deemter plots, which relate the column plate height to the linear mobile phase velocity. A more recent approach to characterize columns is the representation of the peak widths (or the right and left peak half-widths) for a set of compounds versus their retention times, which, for isocratic elution, give rise to almost linear plots. In previous work, these plots have been shown to facilitate the prediction of peak profiles (width and asymmetry) with optimization purposes. In this work, a detailed study on the dependence of the peak widths (or half-widths) on the flow rate is reported. A new approach to quantify the deterioration of column performance for slow and fast flow rates and to characterize chromatographic columns is proposed. The approach makes use of the width (or half-widths) for a set of compounds with similar interaction kinetics and does not require knowledge of the extra-column contributions to the total variance. The chromatographic data of two sets of compounds of different natures (sulfonamides and β-blockers), eluted from Spherisorb and Chromolith columns with acetonitrile-water mixtures, are used to illustrate the approach.

Polyethylenimine modified poly(ethylene terephthalate) capillary channeled-polymer fibers for anion exchange chromatography of proteins

Native poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers have been previously studied as stationary phases for reversed phase and affinity protein separations. In this study, surface modified PET C-CP fibers were evaluated for the anion exchange separation of proteins. The native PET C-CP fibers were aminated using polyethylenimine (PEI) followed by a 1,4-butanediol diglycidyl ether (BUDGE) cross-linking step. Subsequent PEI/BUDGE treatments can be employed to further develop the polyamine layer on the fiber surfaces. The PEI densities of the modified fibers were quantified through the ninhydrin reaction, yielding values of 0.43–0.89μmolg−1. The surface modification impact on column permeability was found to be 0.66×10−11 to 1.33×10−11m2, depending on the modification time and conditions. The dynamic binding capacities of the modified fiber media were determined to be 1.99–8.54mgmL−1 bed volume, at linear velocities of 88–438cmmin−1 using bovine serum albumin as the model protein. It was found that increasing the mobile phase linear velocity (up to 438cmmin−1) had no effect on the separation quality for a synthetic protein mixture, reflecting the lack of van Deemter C-term effects for the C-CP fiber phase. The low-cost, easy modification method and the capability of fast protein separation illustrate great potential in the use of PEI/BUDGE-modified PET C-CP fibers for high-throughput protein separation and downstream processing.

Deductive inference on the plate height equation of ultra performance liquid chromatography

In recent years, separation and analysis workers have made considerable progress on ultra performance liquid chromatography (UPLC). A lot of separation works have been completed which have never been done before. But the theory about chromatographic dynamics of UPLC from the beginning up to now, is remaining in 1950's. Some people explain the principle of UPLC by the van Deemter equation, but this is not right. In this paper, the dynamic process of UPLC has been studied. According to the chromatographic dynamics principle and starting from the heat conduction equation, the plate height equation of UPLC including the influence of mobile phase friction heat production has been deduced as follows: [Formula: see text]. The last item on the right represents the contribution of mobile phase friction heat. When the linear velocity of mobile phase is lower, the contribution of mobile phase friction heat tending to become zero, the plate height equation is reduced to the Horvath and Lin equation. When the linear velocity of mobile phase is high, the temperature difference between the column axial center and edge is directly proportional to the mobile phase linear speed square, and the contribution of thermal effect will largely increased. In this paper, the author clearly pointed out that the column efficiency of UPLC has a direct bearing on the column diameter. Using fine diameter column is helpful to implement column efficiency. Too high mobile phase velocity will lead to the column efficiency collapsed.

Application of core–shell technology for determination of retinol and alpha-tocopherol in breast milk

Breast milk is a main source of fat-soluble vitamins for newborns and it is needful to monitor the nutritional status prior to its application. In this work a novel, high-throughput and low-cost method for monitoring of retinol and alpha-tocopherol in breast milk was developed, validated and compared with reference method using monolithic column.For this purpose five various porous shell and monolithic columns were tested on the basis of relationship between HETP and linear mobile phase velocity, analysis time and consumption of solvents. Finally the core–shell analytical column Kinetex C18 (2.6μm, 100Å, 100×4.6mm) was chosen as the best and optimal values of flow rate, injection volume and temperature of analysis were established.The detection of retinol and alpha-tocopherol was carried out at 325 and 295nm, respectively by diode array detector. The LOD 0.004μmol/L and 0.078μmol/L, the LOQ 0.012μmol/L and 0.182μmol/L for retinol and alpha-tocopherol, respectively were calculated. The validation data showed good linearity, repeatability of retention time with RSD 0.22% and 0.12%, repeatability of peak area with RSD 6.94% and 1.75%, recovery 114.1–116.3% and 99.0–108.6% for retinol and alpha-tocopherol, respectively. Moreover, the newly developed method substantially decreased the solvent consumption by about 263mL per 100 samples with the total time of analysis 1.75min in comparison with analysis time 1.80 of the reference method.

Enantioselective ultra-high and high performance liquid chromatography: A comparative study of columns based on the Whelk-O1 selector

This paper reports on the thermodynamic and kinetic evaluation of a new ultra-high performance liquid chromatography broad-spectrum Pirkle-type chiral stationary phase (CSP) for enantioselective applications (eUHPLC). The well-known Whelk-O1 selector was covalently immobilized onto 1.7-μm high-surface-area, porous spherical silica particles to produce a totally synthetic, covalently bonded CSP that was packed into 150mm, 100mm, 75mm and 50mm columns, either 4.6 or 3.0mm ID. A 100mm×4.6mm ID column was fully characterized from a kinetic and thermodynamic point of view, using as reference a conventional HPLC Whelk-O1 column, 250mm×4.6mm ID, based on 5-μm porous silica particles. On the eUHPLC column, van Deemter plots generated Hmin values of 3.53μm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (μinter) of 5.07mm/s, and Hmin of 4.26 and 4.17μm for the two enantiomers of acenaphthenol, at μinter of 4.85mm/s and 4.24mm/s, respectively. Resolution of 21 enantiomeric pairs including alcohols, epoxides, sulfoxides, phosphine oxides, benzodiazepines and 2-aryloxyproprionic esters used as herbicides, were obtained with significant advantages in terms of efficiency and analysis time. Speed gain factors were calculated for the different column geometries (150mm, 100mm, 75mm and 50mm, either 4.6 or 3.0mm ID), with respect to the standard HPLC column (250mm×4.6mm ID), and were as high as 13, in the case of the 50-mm-long column, affording sub-minute separations of enantiomers with excellent resolution factors. In particular, trans-stilbene oxide was resolved in only 10s, while a 50mm×3.0mm ID column was used as a compromise between reduced mobile phase consumption (less than 1mL per analysis) and smaller extra-column band-broadening effect. Given the relatively low viscosity in NP mode, and the excellent permeability of these eUHPLC columns, with backpressure values under 600bar for a wide range of flow rates, the use of standard HPLC hardware is possible. In this case, however, a significant loss in resolution is observed, compared to the UHPLC instrumentation, if no modifications are introduced in the HPLC apparatus to reduce extra-column variance. The excellent efficiency and selectivity, conjugated with the very high-throughput and the ultra-fast analysis time, prove the potentials of the eUHPLC Whelk-O1 columns in the development of enantioselective UHPLC methods.

Preparation and Evaluation of Monolithic Silica Capillary Column for Reversed-Phase Capillary Liquid Chromatography

Octadecyl-functionalized monolithic silica capillary columns with inner diameters of 100 and 200 micrometers for reversed-phase capillary liquid chromatography were prepared in the fused-silica capillary via sol-gel process. Their performance was evaluated and compared by the separation of alkylbenzenes (toluene, ethylbenzene, propylbenzene, butylbenzene). The column back pressure and linear velocity of mobile phase were investigated. The results show that the monolithic silica capillary columns exhibit good performance for the separation of alkylbenzenes.

Towards porous polymer monoliths for the efficient, retention-independent performance in the isocratic separation of small molecules by means of nano-liquid chromatography

We have investigated the free-radical copolymerization dynamics of styrene and divinylbenzene in the presence of micro- and macro-porogenic diluents in 100 μm I.D. sized molds under conditions of slow thermal initiation leading to (macro)porous poly(styrene- co-divinylbenzene) monolithic scaffolds. These specifically designed experiments allowed the quantitative determination of monomer specific conversion against polymerization time to derive the porous polymer scaffold composition at each desirable copolymerization stage after phase separation. This was carried out over a time scale of 3 h up to 48 h polymerization time, enabling the efficient and repeatable termination of the polymerization reactions. In parallel, the porous and hydrodynamic properties of the derived monolithic columns were thoroughly studied in isocratic nano-LC mode for the reversed-phase separation of a homologous series of small retained molecules. At the optimized initiator concentration, polymerization temperature and time, the macroporous poly(styrene- co-divinylbenzene) monoliths show a permanent mesoporous pore space, which was readily observable by electron microscopy and indicated by nitrogen adsorption experiments. Under these conditions, we consistently find a polymer scaffold composition which suggests a high degree of cross-linking and thus minimum amount of gel porosity. These columns reveal a retention-insensitive plate height in the separation of small retained molecules which only slightly decreases at increased linear mobile phase velocity. As the polymerization progresses, a build-up of less-densely cross-linked material occurs, which is directly reflected in the observed consistent increase in retention and plate heights. This leads to a significant deterioration in overall isocratic separation performance. The decrease in performance is ascribed in particular to the increased mass transfer resistance governing the monoliths’ performance over the whole linear chromatographic flow velocity range at polymerization times significantly higher than that of phase separation. The performance of the optimized monoliths only becomes limited by fluid dispersion due to the poorly structured macroporous pore space.

Density gradients in packed columns: I. Effects of density gradients on retention and separation speed

Density gradients in packed capillary columns operating under the extreme pressure drops typical for solvating gas chromatography were investigated by on-column spectroscopic measurements and compared to a theoretical model. Laser-induced fluorescence was used to follow the elution of various analytes, and Raman spectroscopy was used to measure the density of the mobile phase, each with respect to column position. Mobile phase linear velocity initially increases gradually, and then rises rapidly near the column outlet. High flow rates near the column outlet are offset by a loss of mobile phase solvating power which ultimately limits the speed of separation. These results represent an extreme case for illuminating factors affecting supercritical fluid separation techniques in general.

Nylon-6 capillary-channeled polymer (C-CP) fibers as a hydrophobic interaction chromatography stationary phase for the separation of proteins

Nylon-6 capillary-channeled polymer (C-CP) fibers are used as the stationary phase for the hydrophobic interaction chromatography (HIC) separation of a synthetic protein mixture composed of ribonuclease A, lysozyme, and holotransferrin. Nylon is a useful polymer phase for HIC as it has an alkyl backbone, while the amide functionality is hydrophilic (in fact ionic) in nature. The combination of a nonporous polymer surface of the fiber phases and high column permeability yields very efficient mass transfer characteristics, as exhibited by narrowing of peak widths with increases in mobile phase linear velocity. Retention factors and resolution were evaluated at flow rates ranging from 0.5 to 9 mL/min (linear velocities of ca. 2 to 15 mm/s) and at gradient slopes between 3.3 and 20 %B/min. Optimum resolution was achieved by employing fast flow rates (9 mL/min) and slow gradients (3 %B/min), also resulting in the highest peak capacities.

Single‐step preparation and characterization of polymeric monolith for pressurized capillary electrochromatography of typical homologs

A monolithic stationary phase was prepared in a single step by in situ copolymerization of iso-butyl methacrylate (IBMA), ethylene dimethacrylate (EDMA), and N,N-dimethylallylamine (DMAA) in a binary porogenic solvent consisting of N,N-dimethylformamide (DMF) and 1,4-butanediol. As the frame structures of monoliths, the amino groups are linked to support the EOF necessary for driving the mobile phase through the monolithic capillary, while the hydrophobic groups are introduced to provide the nonpolar sites for the chromatographic retention. To evaluate the column performance, separations of typical kinds of neutral or charged homologs, such as alkylbenzenes, phenols (including isomeric compounds of hydroquinone, resorcin, and catechol), and anilines (including isomeric compounds of o-phenylenediamine and 1,4-phenylenediamine), were performed, respectively on the prepared column under the mode of pressurized pCEC. Effects of the buffer pH and the mobile phase composition on the linear velocity of mobile phase and the retention factors of these compounds were investigated. It was found that the retention mechanism of charged solutes could be attributed to a mixed mode of hydrophobic interaction and electrophoresis, while an RP chromatographic behavior on the monolithic stationary phases was exhibited for neutral solutes. Especially, basic compounds such as anilines were well separated on the monolithic columns in the "counterdirectional mode," which effectively eliminated the electrostatic adsorption of basic analytes on the charged surface of the stationary phases.

The application of small porous particles, high temperatures, and high pressures to generate very high resolution LC and LC/MS separations

The effect of combining sub-2 microm porous particles with elevated operating temperatures on chromatographic performance has been investigated in terms of chromatographic efficiency, productivity, peak elution order, and observed operating pressure. The use of elevated temperature in LC does not increase the obtainable performance but allows the same performance to be obtained in less time. Increasing the column temperature did allow the use of longer columns, generating column efficiencies in excess of 100,000 plates and gradient peak capacities approaching 1000. Raising the temperature increased the optimal mobile phase linear velocity, negating somewhat the pressure benefits observed by reducing the solvent viscosity. When operating at higher temperature the analyte retention is not only reduced, but the order of elution will also often change. High temperature separations allowed exotic organic modifiers such as isopropanol to be exploited for alternative selectivity and faster analysis. Finally, care must be taken when using high temperature separations to ensure that the narrow peak widths produced do not compromise the quality of data obtained from detectors such as high resolution mass spectrometers.

Performance of octadecylsilylated monolithic silica capillary columns of 530 μm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.

Enhanced Sequence Coverage in Tryptic Fragment Analysis by Two-Dimensional HPLC/MS Using a Monolithic Silica Capillary Column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.